1. Field of the Invention
The present invention relates generally to the field of immunology. More particularly, it concerns the causal role for diacylglycerol kinase (DGK) and defective Ras signaling in T cell anergy. An implementation of the invention relates to the identification of compounds that can modulate or mimic DGK-α and/or DGK-ζ activity and the use of these compounds to treat conditions that result from immune dysregulation.
2. Description of the Related Art
Engagement of the T cell receptor for antigen (TCR) in the absence of CD28 costimulation can result in a long-term hyporesponsive state termed clonal anergy (Schwartz, 2003). Anergic T cells show defective IL-2 production and proliferation upon restimulation via the TCR and CD28, and produce other cytokines at reduced levels. Anergy may represent one mechanism of peripheral tolerance (Ramsdell et al., 1989), and has been reported to occur in the setting of non-productive anti-tumor immunity in vivo (Staveley-O'Carroll et al., 1998). Thus, understanding the regulation of induced T cell hyporesponsiveness may enable manipulation of immune responses to favor tolerance versus activation, having broad potential application toward disease states associated with immune dysregulation.
The molecular alterations in the anergic state that correlate with defective IL-2 production in response to TCR/CD28 engagement have been evaluated in some detail (Fields et al., 1996). The most compelling correlative signaling perturbation is a deficiency in Ras activation, which is associated with diminished activation of the MAP kinases ERK and JNK (Fields et al., 1996; Li et al., 1996) as well as blunted AP-1 transactivation (Kang et al., 1992). The ability of dominant negative (DN) Ras to inhibit IL-2 promoter activity in T cell tumor lines (Rayter et al., 1992) coupled with the block in thymic development seen in DN Ras transgenic mice (Swan et al., 1995) support an important role for Ras signaling in TCR-mediated T cell activation. Nevertheless, whether deficient Ras activation is sufficient to explain the anergic phenotype has not been formally demonstrated.
In part, the lack of mechanistic data regarding Ras and anergy is due to the technical limitations of current strategies for genetic manipulation of normal T cells in vitro. In particular, a key study would require introduction of constitutively active (CA) Ras into already anergized T cells, to determine if MAP kinase activation and IL-2 production could be restored. Retroviral transduction would require cell proliferation for integration and gene expression, which is not possible with growth-arrested anergic T cells. However, by utilizing T cells from Coxsackie and adenovirus receptor (CAR) transgenic mice (Wan et al., 2000), a model which enables adenoviral transduction of quiescent non-proliferating cells, the inventor shows that that introduction of CA Ras61L into resting anergic T cells restores IL-2 production and MAP kinase activation, indicating a causal role for blocked Ras in mediating the anergic state. The results are in apparent contrast to a recent report in which T cells transduced with a retrovirus encoding active Ras were still rendered hyporesponsive (Crespi et al., 2002). However, retroviral transduction required that the cells be transduced first and then anergized, and it is conceivable that early and constitutive Ras activation might itself contribute to T cell dysregulation. The experimental approach used by the inventor allowed active Ras to be introduced after anergy was induced, and in model systems in which a blockade in Ras signaling is known to occur.
Previous reports have suggested that increased recruitment of CrkL-C3G complexes and concomitant Rap1 activation might be responsible for the decreased TCR-induced Ras/MAP kinase activation and IL-2 production in anergic cells (Boussiotis et al., 1997). However, the inventor recently observed that CrkL-deficient mice generated by gene targeting do not show T cell hyperresponsiveness (Peterson et al., 2003), and the present invention shows that Th1 cells derived from CrkL-deficient mice are still capable of being anergized. In addition, recent work has shown that transgenic expression of Rap1 in T cells promotes increased T cell activation through augmented adhesion (Sebzda et al., 2002). Collectively, these results make it unlikely that CrkL-C3G and Rap1 are involved in a major negative regulatory pathway in T cells, although these molecules could conceivably dampen TCR signaling under other circumstances.
Other gene products have been reported as candidates for contributing to T cell hyporesponsiveness in the anergic state. The transcriptional regulator Tob has been found to be upregulated in anergic cells, and transfection of cells to overexpress Tob led to diminished IL-2 production (Tzachanis et al., 2001). However, Tob was also found to be highly expressed in naive T cells, and naive T cells show robust IL-2 production in response to TCR/CD28 ligation. Thus, although signals involving Tob may be important for the negative regulation of T cell activation, they do not appear to parallel the anergic phenotype. A recent report has identified GRAIL, an E3 ubiquitin ligase, as being upregulated in anergic cells and negatively regulating TCR-mediated cytokine gene expression (Anandasabapathy et al., 2003). It is conceivable that GRAIL could also antagonize Ras signaling, and/or that diminished Ras signaling could promote GRAIL upregulation.
Although previous studies correlated T cell anergy with defective Ras signaling, neither a causal relationship nor the mechanism of Ras hypoactivation had been established. Thus, a better understanding of the regulation of induced T cell hyporesponsiveness is needed to enable manipulation of immune responses to favor tolerance versus activation. The ability to manipulate the immune response would have the potential for broad application toward disease states associated with immune dysregulation.